The bacteriophage λ Red homologous recombination system has been studied over the past 50 years as a model system to define the mechanistic details of how organisms exchange DNA segments that share extended regions of homology. The λ Red system proved useful as a system to study because recombinants could be easily generated by co-infection of genetically marked phages. What emerged from these studies was the recognition that replication of phage DNA was required for substantial Red-promoted recombination in vivo, and the critical role that double-stranded DNA ends play in allowing the Red proteins access to the phage DNA chromosomes. In the past 16 years, however, the λ Red recombination system has gained a new notoriety. When expressed independently of other λ functions, the Red system is able to promote recombination of linear DNA containing limited regions of homology (∼50 bp) with the Escherichia coli chromosome, a process known as recombineering. This review explains how the Red system works during a phage infection, and how it is utilized to make chromosomal modifications of E. coli with such efficiency that it changed the nature and number of genetic manipulations possible, leading to advances in bacterial genomics, metabolic engineering, and eukaryotic genetics.

All cells must accurately replicate DNA and partition it to daughter cells. The basic cell cycle machinery is highly conserved among eukaryotes. Most of the mechanisms that control the cell cycle were worked out in fungal cells, taking advantage of their powerful genetics and rapid duplication times. Here we describe the cell cycles of the unicellular budding yeast Saccharomyces cerevisiae and the multicellular filamentous fungus Aspergillus nidulans. We compare and contrast morphological landmarks of G1, S, G2, and M phases, molecular mechanisms that drive cell cycle progression, and checkpoints in these model unicellular and multicellular fungal systems.

Synthetic DNA is looking increasingly promising as a compact means for storing and retrieving different types of data, including text and images, according to Luis Ceze and Georg Seelig at the University of Washington, Doug Carmean and Karin Strauss of Microsoft Research, also in Seattle, and their collaborators. Their approach to exploiting DNA for data storage offers improved “controllable redundancy, reliability, and [information-storage] density” over previous attempts and is one of the first systems that uses DNA molecules to store digital images and retrieve them intact, they say. Details were presented last April in Atlanta during the annual Association for Computing Machinery International Conference on Architectural Support for Programming Languages and Operating Systems (https://homes.cs.washington.edu/luisceze/publications/dnastorage-asplos16.pdf).

Through some unexpected and still-unidentified form of molecular texting, the herpes simplex virus (HSV) induces a hormone-like stimulation of cellular DNA replication in nearby, uninfected cells, according to Peter O'Hare of St. Mary's Medical School, Imperial College in London, United Kingdom, and his collaborators. Details appeared 26 August 2015 in the Journal of Virology (doi:10.1128/JVI.01950–15/).

DNA topoisomerases are enzymes that control the topology of DNA in all cells. There are two types, I and II, classified according to whether they make transient single- or double-stranded breaks in DNA. Their reactions generally involve the passage of a single- or double-strand segment of DNA through this transient break, stabilized by DNA-protein covalent bonds. All topoisomerases can relax DNA, but DNA gyrase, present in all bacteria, can also introduce supercoils into DNA. Because of their essentiality in all cells and the fact that their reactions proceed via DNA breaks, topoisomerases have become important drug targets; the bacterial enzymes are key targets for antibacterial agents. This article discusses the structure and mechanism of topoisomerases and their roles in the bacterial cell. Targeting of the bacterial topoisomerases by inhibitors, including antibiotics in clinical use, is also discussed.

Recent national and international developments involving microbiology and related science policy matters include:

Reflecting a continuing concern over antibiotic resistance issues, the Obama administration in June invited representatives from more than 150 food companies, retailers, and human and animal health stakeholders to a “White House Forum on Antibiotic Stewardship.” The President also signed a memorandum directing federal departments and agencies to create “a preference for meat and poultry produced according to responsible antibiotic use.”

In a related development, officials of the Food and Drug Administration plan to expand data collection on the use of antibiotics in agriculture.

Further, the National Institutes of Health and the Biomedical Advanced Research and Development Authority in June announced a competition in which up to $20 million could be made available for the delivery of one or more successful rapid point-of-care diagnostics that may be used by health care providers to identify bacterial infections.

The World Health Assembly (WHA) in May adopted a global action plan on antimicrobial resistance, urging member states to implement compatible national action plans within two years.

WHA also called on member nations to allocate adequate resources for introducing vaccines and developing immunization programs.

Back at the national level, several members of the House of Representatives Energy and Commerce Committee are continuing to question Centers for Disease Control and Prevention Director Tom Frieden and Secretary of Defense Ashton Carter about incidents in which samples containing “live anthrax” were mistakenly shipped “to nearly twice as many states and three times as many countries as originally reported, [including] 51 labs in 17 states and 3 foreign countries,” the committee members note.

The U.S. Department of Energy (DOE) Joint Genome Institute (JGI) was established in 1997 to consolidate the department's programs and resources in DNA sequencing, informatics, and technology development. Soon after, the University of California, which manages the Lawrence Berkeley National Laboratory, where JGI was first situated, leased lab and office space in nearby Walnut Creek to house JGI activities. The early focus for JGI was the Human Genome Project. After its scientists completed their sequencing of three human chromosomes, however, JGI broadened its mandate in 2004 to become a national user facility, which now boasts thousands of users worldwide.

The highly conserved Nus factors of bacteria were discovered as essential host proteins for the growth of temperate phage λ in Escherichia coli. Later, their essentiality and functions in transcription, translation, and, more recently, in DNA repair have been elucidated. Close involvement of these factors in various gene networks and circuits is also emerging from recent genomic studies. We have described a detailed overview of their biochemistry, structures, and various cellular functions, as well as their interactions with other macromolecules. Towards the end, we have envisaged different uncharted areas of studies with these factors, including their participation in pathogenicity.

The DNA of Escherichia coli contains 19,120 6-methyladenines and 12,045 5-methylcytosines in addition to the four regular bases, and these are formed by the postreplicative action of three DNA methyltransferases. The majority of the methylated bases are formed by the Dam and Dcm methyltransferases encoded by the dam (DNA adenine methyltransferase) and dcm (DNA cytosine methyltransferase) genes. Although not essential, Dam methylation is important for strand discrimination during the repair of replication errors, controlling the frequency of initiation of chromosome replication at oriC, and the regulation of transcription initiation at promoters containing GATC sequences. In contrast, there is no known function for Dcm methylation, although Dcm recognition sites constitute sequence motifs for Very Short Patch repair of T/G base mismatches. In certain bacteria (e.g., Vibrio cholerae, Caulobacter crescentus) adenine methylation is essential, and, in C. crescentus, it is important for temporal gene expression, which, in turn, is required for coordinating chromosome initiation, replication, and division. In practical terms, Dam and Dcm methylation can inhibit restriction enzyme cleavage, decrease transformation frequency in certain bacteria, and decrease the stability of short direct repeats and are necessary for site-directed mutagenesis and to probe eukaryotic structure and function.

The purpose of this essay is threefold: to give an outline of the life and the various achievements of Theodor Escherich, to provide a background to his discovery of what he called Bacterium coli commune (now Escherichia coli), and to indicate the enormous impact of studies with this organism, long before it became the cornerstone of research in bacteriology and in molecular biology.

Students design primers using web-based tools along with a printed sequence of a prokaryotic gene showing both DNA strands and translation of the coding strand into amino acids. The melting temperature of each primer is determined and the pair closest in temperature can be used to amplify and clone a gene into a vector for a one-semester laboratory exercise. By working through this primer design exercise, students see first hand what is involved in the process of amplifying DNA.

Cellular DNA is constantly challenged by various endogenous and exogenous genotoxic factors that inevitably lead to DNA damage: structural and chemical modifications of primary DNA sequence. These DNA lesions are either cytotoxic, because they block DNA replication and transcription, or mutagenic due to the miscoding nature of the DNA modifications, or both, and are believed to contribute to cell lethality and mutagenesis. Studies on DNA repair in Escherichia coli spearheaded formulation of principal strategies to counteract DNA damage and mutagenesis, such as: direct lesion reversal, DNA excision repair, mismatch and recombinational repair and genotoxic stress signalling pathways. These DNA repair pathways are universal among cellular organisms. Mechanistic principles used for each repair strategies are fundamentally different. Direct lesion reversal removes DNA damage without need for excision and de novo DNA synthesis, whereas DNA excision repair that includes pathways such as base excision, nucleotide excision, alternative excision and mismatch repair, proceeds through phosphodiester bond breakage, de novo DNA synthesis and ligation. Cell signalling systems, such as adaptive and oxidative stress responses, although not DNA repair pathways per se, are nevertheless essential to counteract DNA damage and mutagenesis. The present review focuses on the nature of DNA damage, direct lesion reversal, DNA excision repair pathways and adaptive and oxidative stress responses in E. coli.

All living organisms are continually exposed to agents that damage their DNA, which threatens the integrity of their genome. As a consequence, cells are equipped with a plethora of DNA repair enzymes to remove the damaged DNA. Unfortunately, situations nevertheless arise where lesions persist, and these lesions block the progression of the cell's replicase. In these situations, cells are forced to choose between recombination-mediated "damage avoidance" pathways or a specialized DNA polymerase (pol) to traverse the blocking lesion. The latter process is referred to as Translesion DNA Synthesis (TLS). As inferred by its name, TLS not only results in bases being (mis)incorporated opposite DNA lesions but also bases being (mis)incorporated downstream of the replicase-blocking lesion, so as to ensure continued genome duplication and cell survival. Escherichia coli and Salmonella typhimurium possess five DNA polymerases, and while all have been shown to facilitate TLS under certain experimental conditions, it is clear that the LexA-regulated and damage-inducible pols II, IV, and V perform the vast majority of TLS under physiological conditions. Pol V can traverse a wide range of DNA lesions and performs the bulk of mutagenic TLS, whereas pol II and pol IV appear to be more specialized TLS polymerases.

Early research on the origins and mechanisms of mutation led to the establishment of the dogma that, in the absence of external forces, spontaneous mutation rates are constant. However, recent results from a variety of experimental systems suggest that mutation rates can increase in response to selective pressures. This chapter summarizes data demonstrating that,under stressful conditions, Escherichia coli and Salmonella can increase the likelihood of beneficial mutations by modulating their potential for genetic change.Several experimental systems used to study stress-induced mutagenesis are discussed, with special emphasison the Foster-Cairns system for "adaptive mutation" in E. coli and Salmonella. Examples from other model systems are given to illustrate that stress-induced mutagenesis is a natural and general phenomenon that is not confined to enteric bacteria. Finally, some of the controversy in the field of stress-induced mutagenesis is summarized and discussed, and a perspective on the current state of the field is provided.

Homologous recombination is an ubiquitous process that shapes genomes and repairs DNA damage. The reaction is classically divided into three phases: presynaptic, synaptic, and postsynaptic. In Escherichia coli, the presynaptic phase involves either RecBCD or RecFOR proteins, which act on DNA double-stranded ends and DNA single-stranded gaps, respectively; the central synaptic steps are catalyzed by the ubiquitous DNA-binding protein RecA; and the postsynaptic phase involves either RuvABC or RecG proteins, which catalyze branch-migration and, in the case of RuvABC, the cleavage of Holliday junctions. Here, we review the biochemical properties of these molecular machines and analyze how, in light of these properties, the phenotypes of null mutants allow us to define their biological function(s). The consequences of point mutations on the biochemical properties of recombination enzymes and on cell phenotypes help refine the molecular mechanisms of action and the biological roles of recombination proteins. Given the high level of conservation of key proteins like RecA and the conservation of the principles of action of all recombination proteins, the deep knowledge acquired during decades of studies of homologous recombination in bacteria is the foundation of our present understanding of the processes that govern genome stability and evolution in all living organisms.

This chapter describes DNA repair systems that have not been described for Candida species even though orthologues are found at least in the Candida albicans genome databases. A section of the chapter describes the genetic plasticity as it relates to drug resistance. In Saccharomyces cerevisiae haploid cells, the rate of spontaneous mutation in the nuclear genome is rather low under laboratory conditions. An additional marker of the genetic instability in C. albicans is represented by aneuploidies. Aneuploidies are common in laboratory strains of C. albicans but are especially abundant when those strains have been subjected to genetic manipulations, including several laboratory strains successively derived from CAI-4, or treated with mutagenic agents such as UV light. Genetic instability could be caused by an increase in the rate of mutations in the form of single base substitutions, microinsertions, and microdeletions. These alterations are known to arise from errors during normal DNA replication by polymerases δ and ε and are usually corrected before being fixed by methyl mismatch repair (MMR). It was suggested that C. albicans has evolved additional DNA repair systems to defend itself against killing by the oxygen radicals generated by macrophages. For an opportunistic pathogen, drug resistance represents an excellent and practical system to correlate phenotypic traits with genomic changes. Azoles are drugs commonly used in clinics. Hypermutable subpopulations are characterized by the presence of secondary mutations unrelated to that selected, which are distributed throughout the genome.

This chapter describes the major Candida albicans morphologies and the current understanding of the cell biological and cell cycle features that distinguish them. It highlights recent insights into how cell cycle regulators influence the formation of hypha-specific cellular features in particular. Since morphogenesis and cell cycle regulation have been studied most extensively in C. albicans, the chapter primarily focuses on work in C. albicans. The important distinction between yeast and pseudohyphae is that pseudohyphae spend more time in G2 phase of the cell cycle than yeast cells , and they continue to elongate during this time. There has long been a controversy as to how pseudohyphae are related to true hyphae. Initial models suggested that yeast cells, pseudohyphae, and true hyphae reside along a continuum. Later, based on differences in cell cycle dynamics and subcellular structures, it was proposed that pseudohyphae and hyphae represent two distinct morphological states, with pseudohyphae being more like yeast form growth with respect to cell cycle progression and cell biological markers. Recent work has shed light on cell biological features associated with cell cycle progression in chlamydospores and is discussed in theis chapter. In the C. albicans genome sequence, there are three G1 cyclins (Ccn1, Cln3, and Hgc1) and two G2 or B-type/mitotic cyclins (Clb2 and Clb4) that are predicted to associate with Cdc28.

One of the major emphases of the author's research program is to understand how obligate intracytoplasmic growth has affected the physiology of Rickettsia prowazekii. This chapter discusses metabolism and reductive evolution from the pathogenic rickettsia's point of view. Rapid advances in sequencing technologies have contributed to the ever-expanding availability of genome sequence information. This has significantly augmented our understanding of the factors that influence virulence and shape pathogen evolution at the genome level. The chapter summarizes the studies describing rickettsial physiology and metabolism before 1998, when the first rickettsial genome sequence became available. It provides insight into some of the key experiments that guided the field during a productive period in rickettsial research. The R.prowazekii adenosine triphosphate (ATP)/adenosine diphosphate (ADP) translocase is the best-characterized rickettsial transport system. It is well established that the ATP/ADP translocase functions via an obligate exchange antiport mechanism and thus requires the presence of substrate on both sides of the membrane to catalyze transport. Studies examining the physiology of rickettsiae that are growing intracellularly have contributed much to the understanding of rickettsia-host interactions. The chapter discusses how obligate intracellular growth has affected the rickettsia's capacity for gene regulation. As a final facet of rickettsial gene regulation, transcriptional termination is also explained in the chapter.

Many bacterial regulatory circuits are not digital but analogue devices. In other words, their responses are not all-or-none but are proportional to the stimulus to which they respond. Overlap and redundancy contribute to bacterial robustness. Bacterial populations benefit from nondeterministic, random variations in their molecular circuitry-called epigenetic variation because the processes are heritable but not due to mutational changes in the DNA. Extrachromosomal DNA has been traditionally viewed as the main toolbox for bacterial genome plasticity. Plasmids are crucial indeed for bacterial adaptation. Studies with clinical isolates have indicated that hypermutable bacterial lineages may adapt better to harsh environmental conditions. While it seems out of the question that such hypermutable lineages may enter an evolutionary dead end, their existence emphasizes the importance of mutation as an adaptive strategy. Population geneticists have predicted that variation of mutation rates in response to environmental circumstances might have selective value. Under comfortable circumstances, however, elevated mutation rates would be unnecessary and probably detrimental. An example of variation of mutation rates upon environmental influence is observed when E. coli is exposed to fluoroquinolones, a class of antibiotics that target DNA topoisomerases, thus blocking DNA replication. Increased mutation rates may produce additional mutations that can further facilitate survival. SOS induction associated with antibiotic challenge is not the only environmentally controlled mechanism known to modulate mutation rates. Bacteria are equipped with analogue devices that permit efficient adaptation to changing conditions. Bacterial populations often display bistable or multistable states, created either by built-in mechanisms or by random fluctuations.

DNA exonucleases, enzymes that hydrolyze phosphodiester bonds in DNA from a free end, play important cellular roles in DNA repair, genetic recombination and mutation avoidance in all organisms. This article reviews the structure, biochemistry, and biological functions of the 17 exonucleases currently identified in the bacterium Escherichia coli. These include the exonucleases associated with DNA polymerases I (polA), II (polB), and III (dnaQ/mutD); Exonucleases I (xonA/sbcB), III (xthA), IV, VII (xseAB), IX (xni/xgdG), and X (exoX); the RecBCD, RecJ, and RecE exonucleases; SbcCD endo/exonucleases; the DNA exonuclease activities of RNase T (rnt) and Endonuclease IV (nfo); and TatD. These enzymes are diverse in terms of substrate specificity and biochemical properties and have specialized biological roles. Most of these enzymes fall into structural families with characteristic sequence motifs, and members of many of these families can be found in all domains of life.